Spermatozoa Develop Molecular Machinery to Recover From Acute Stress

This study was designed to search for the possible mechanism(s) of male (in/sub)fertility by following the molecular response of spermatozoa on acute psychological stress (the most common stress in human society) and on a 20-h time-dependent recovery period. To mimic in vivo acute stress, the rats were exposed to immobilization once every 3 h. The recovery periods were as follows: 0 (immediately after stress and 3 h after the light is on—ZT3), 8 (ZT11), 14 (ZT17), and 20 (ZT23) h after stress. Results showed that acute stress provoked effects evident 20 h after the end of the stress period. Numbers of spermatozoa declined at ZT17 and ZT23, while functionality decreased at ZT3 and ZT11, but recovered at ZT17 and ZT23. Transcriptional profiles of 91% (20/22) of tracked mitochondrial dynamics and functionality markers and 91% (20/22) of signaling molecules regulating both mitochondrial dynamics and spermatozoa number/functionality were disturbed after acute stress and during the recovery period. Most of the changes presented as increased transcription or protein expression at ZT23. The results of the principal component analysis (PCA) showed the clear separation of acute stress recovery effects during active/dark and inactive/light phases. The physiological relevance of these results is the recovered positive-acrosome-reaction, suggesting that molecular events are an adaptive mechanism, regulated by acute stress response signaling. The results of the PCA confirmed the separation of the effects of acute stress recovery on gene expression related to mitochondrial dynamics, cAMP, and MAPK signaling. The transcriptional patterns were different during the active and inactive phases. Most of the transcripts were highly expressed during the active phase, which is expected given that stress occurred at the beginning of the inactive phase. To the best of our knowledge, our results provide a completely new view and the first presentation of the markers of mitochondrial dynamics network in spermatozoa and their correlation with signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality during recovery from acute stress. Moreover, the interactions between the proteins important for spermatozoa homeostasis and functionality (MFN2 and PRKA catalytic subunit, MFN2 and p38MAPK) are shown for the first time. Since the existing literature suggests the importance of semen quality and male fertility not only as the fundamental marker of reproductive health but also as the fundamental biomarkers of overall health and harbingers for the development of comorbidity and mortality, we anticipate our result to be a starting point for more investigations considering the mitochondrial dynamics markers or their transcriptional profiles as possible predictors of (in/sub)fertility.


Supplementary
. Transcription of mitochondrial fusion and architecture markers in spermatozoa of acutely stressed adult rats is the most prominently changed at the ZT23 time point. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for analysis of the transcriptional profile of markers of mitochondrial fusion and architecture. Data bars are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at level p <0.05: * vs. control group of the same time point. Figure S3. Transcription of mitochondrial fission markers in spermatozoa of acutely stressed adult rats is the most prominently changed at the ZT23 time point. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for analysis of the transcriptional profile of markers of mitochondrial fission. Data bars are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at level p <0.05: * vs. control group of the same time point. Figure S4. Transcription of mitochondrial autophagy markers in spermatozoa of acutely stressed adult rats is the most prominently changed at the ZT23 time point. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for analysis of the transcriptional profile of markers of mitochondrial autophagy. Data bars are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at level p <0.05: * vs. control group of the same time point. Figure S5. Transcription of mitochondrial functionality markers in spermatozoa of acutely stressed adult rats is the most prominently changed at the ZT23 time point. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for analysis of the transcriptional profile of markers of mitochondrial functionality. Data bars are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at level p <0.05: * vs. control group of the same time point. Figure S6. Transcription of cAMP signaling markers regulating mitochondrial dynamics and functionality as well as spermatozoa number and functionality in spermatozoa of acutely stressed adult rats is the most prominently changed at the ZT23 time point. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for analysis of the transcriptional profile of markers of cAMP signaling pathway. Data bars are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at level p <0.05: * vs. control group of the same time point. Figure S7. Transcription of MAPK signaling markers in spermatozoa of acutely stressed adult rats is the most prominently changed at the ZT23 time point. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for analysis of the transcriptional profile of markers of MAPK signaling pathway. Data bars are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at level p <0.05: * vs. control group of the same time point. Figure S8. Heat map analysis of transcriptional profile of mitochondrial dynamic and functionality markers (A) and signaling molecules regulating mitochondrial dynamics and functionality (B) in spermatozoa of acutely stressed adult rats with recovery at different time points (0hours-ZT3, 8hours-ZT11, 14hours-ZT17, 20hours-ZT23). The relative fold changes in the genes expression for the before mentioned genes were calculated using the corresponding control group of the same time point as a calibrator to show the changes within each single time point (ZT3, ZT11, ZT17 and ZT23). Color from red to green indicates low to high expression.Relative protein expression of mitochondrial biogenesis and functionality markers as well as markers of signaling pathways regulating mitochondrial dynamic and spermatozoa functionality in spermatozoa samples after MFN2 immunoprecipitation analysis Considering the relationship of mitofusin 2 expression and motility and cryoprotective potential of human spermatozoa (1), immunoprecipitation analysis of MFN2 protein was performed in spermatozoa samples of stressed animals with different recovery periods. Sprermatozoa samples for immunoprecipitation analysis were lysed and concentration of proteins in each sample was estimated by Bradford method (set at concentration of 300 µg/ml). Pre-clearing of the lysate was done using normal goat serum (Santa Cruz Biotechnology, normal goat serum: sc-2043, (https://www.scbt.com)). After pre-clearing step, lysates were mixed with MFN2 antibody (Santa Cruz Biotechnology). Immunoprecipitated complexes with MFN2 antibody were recovered by protein G agarose bead slurry and supernatant was used for further protein analysis using Western blot method. Results show that there is no significant difference in protein expression of ERRɑ (Supplemental Figure 9A) and ERK1/2 (Supplemental Figure 9C) proteins in all analyzed groups compared to control group of ZT3 time point. Results show that there is significant decrease in 1x3hIMO+R group of ZT3 time point, and oppositely increase in 1x3hIMO+R group of ZT23 time point in the expression of NRF1 protein in supernatant after the immunoprecipitation of MFN2 protein in spermatozoa (Supplemental figure  9B). Relative expression of p38MAPK protein in supernatant after the immunoprecipitation of MFN2 protein, was decreased in stressed groups with recovery periods of 0, 8, 14 and 20 hours (ZT3, ZT11, ZT17 and ZT23), as well as in control groups of ZT11 and ZT17 time points, compared to control group of ZT3 time point (Supplemental Figure 9D). Since immunoprecipitation analysis show protein interaction between MFN2 and PRKAc, analysis for the PRKAc protein in the supernatant was performed. The results show that there is no PRKAc protein present in the supernatant sample after the immunoprecipitation analysis (Supplemental Figure 9E). Figure S9. Relative expression of ERRɑ, NRF1, ERK1/2 and p38 MAPK proteins as mitochondrial functionality markers as well as markers of signaling pathways regulating mitochondrial dynamic and spermatozoa functionality in spermatozoa samples after MFN2 immunoprecipitation analysis. Relative expression of (A) ERRɑ, (B) NRF1, (C) ERK1/2 and (D) p38 MAPK are analyzed in supernatant after MFN2 immunoprecipitation analysis. Analysis of PRKAc (E) in supernatant after MFN2 immunoprecipitation analysis prove the interaction of the two analysed proteins shown in main manuscript file. Spermatozoa samples for immunoprecipitation analysis were lysed and concentration of proteins in each sample was estimated by Bradford method and set at concentration of 300 µg/ml. Pre-clearing of the lysate was done using normal goat serum. After pre-clearing step, lysates were mixed with MFN2 antibody. Immunoprecipitated complexes with MFN2 antibody were recovered by protein G agarose bead slurry and supernatant was used for further protein analysis using Western blot method. Data bars are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at level p <0.05: # vs. control group of the ZT3 time point, * vs. control group of the same time point.

Mitochondrial membrane potential (∆ψ) measurement using TMRE fluorescent dye in adult capacitated and non-capacitated spermatozoa after one hour ex vivo adrenaline treatment
Three-months-old male Wistar rats were used for the experiment. Adult animals were not subject to any previous treatment. Spermatozoa were isolated from the caudal epididymides and were incubated in capacitated or non-capacitated medium for one hour. After the incubation period, capacitated and non-capacitated spermatozoa were treated ex vivo with adrenaline in dose of 10 µM for 3 hours.
To monitor the membrane potential of mitochondria (∆ψ) in spermatozoa, tetramethylrhodamine ethyl ester (TMRE) as fluorescent probe was employed according to the procedure published previously (2). Briefly, 1x10 5 spermatozoa were loaded in each well of a 96-well black plate, with eight replicates from each group. Spermatozoa in a 96-well plate were left for 3 hours in the incubator at 37 °C, to recover from the stimulation procedure. After that period medium was aspirated, TMRE fluorescent dye was added in a final concentration of 100 nM and incubated for 20 minutes at 37 °C. Spermatozoa were washed from TMRE fluorescent dye by aspiration and fluorescence was measured in 0,1% BSA-1xPBS solution (Ex/Em 550/590 nm). After the measurement, all wells were washed with 1xPBS solution and frozen at -20 °C until the measurement of protein concentration in each well by Bradford protein assay.   Primers were designed by using software Primer Express 3.0 (Applied Biosystems) and full genes sequences from the NCBI Entrez Nucleotide database (www.ncbi.nlm.nih.gov/sites/entrez). F -forward; R -reverse.  Primers were designed by using software Primer Express 3.0 (Applied Biosystems) and full genes sequences from the NCBI Entrez Nucleotide database (www.ncbi.nlm.nih.gov/sites/entrez). F -forward; R -reverse.

Supplementary
Supplementary table S10. Primers sequences used for the real-time PCR analysis of cAMP signaling elements.

Relative quantification of protein expression after immunoprecipitation analysis
Sprermatozoa samples for immunoprecipitation analysis were lysed in 1 ml buffer containing 20 mM HEPES, 10 mM EDTA, 2.5 mM MgCl2, 40 mM β-glycerophosphate, 1mM DTT, 1% NP-40, 0.5 mM 4-(aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1 µM aprotinin, 2 µM leupeptin and Phosphatase inhibitor cocktail tablets (cont. (1R, 2S, 3R, 6S)-1.2-dimethyl-3.6-epoxycyclohexane-1.2-dicarboxylic anhydride). Concentration of proteins in each sample was estimated by Bradford method and set at concentration of 300 µg/ml. Equal amount of protein in each sample (300 µg) was used for the immunoprecipitation. Pre-clearing of the lysate was done using 5 µl of normal goat serum (Santa Cruz Biotechnology, normal goat serum: sc-2043, (https://www.scbt.com)) mixed with 1 ml of lysate and incubated on ice for 1 hour. After the incubation 100 µl of bead slurry was added to each sample and incubated for 30 minutes at 4 °C with gentle agitation. Supernatant for the immunoprecipitation was collected after 10 minutes at 14 000xg at 4 °C centrifugation. After preclearing process, lysates were mixed with MFN2 antibody (Santa Cruz Biotechnology) and incubated at 4 °C overnight with constant rotation. During additional overnight incubation at 4 °C with constant rotation, immunoprecipitated complexes with MFN2 antibody were recovered by 80 µl of protein G agarose bead slurry and supernatant was used for further protein analysis by Western blot. Western blot analysis was done in the same manner as the analysis described in the main manuscript text and as described previously (1). Immune-reactive bands were detected using MyECL Imager (Thermo Fisher Scientific Inc.; https://www.thermofisher.com) and analyzed as two-dimensional images using Image J version 1.48 (http://rsbweb.nih.gov/ij/download.html). The optical density of images is expressed as volume adjusted for the background, which gives arbitrary units of adjusted volume. Normalization of the data was done using ACTIN protein expression, as the endogenous control. Immune-detection was performed with different antibodies (all details are listed in Supplementary  Table S12).